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Mechanisms responsible for oestrogen receptor expression in primary human breast cancer
The Breast (1996) 5.231-243 0 1996 Pearson Professional Ltd
ORIGINAL ARTICLE
Mechanisms responsible for oestrogen receptor expression in primary human breast cancer A. K. Sharma, D. Grimshaw, K. Horgan, R. E. Mansel, J. M. W . Gee* and R. I. Nicholson* Department of Surgery and *Tenovus Cancer Research Centre, University of Wales College of Medicine, Cardifi
UK
S UMMA R Y. Oestrogen receptor (ER) status in human breast cancer provides the best parameter available at present to determine the likely response to endocrine therapy. However, the precise mechanisms responsible for ER expression have yet to be fully elucidated. In order to examine this we have quantitatively analysed the mRNA and protein levels of ER in 61 patients with primary breast cancer using the techniques of ribonucleic acid (RNA) slot blotting and immunocytochemistry. Using the H222 monoclonal antibody, ER staining was observed in 40 cases (65.5%) following immunocytochemical assay. RNA slot blotting using a complimentary deoxyribonucleic acid probe (HEO) revealed ERmRNA to be present in 46 cases (75.4%). The mRNA and protein levels of ER were found to be significantly correlated (rs = 0.753, P < 0.0001). Exclusion of 15 cases where neither mRNA nor protein was detected still revealed a significant correlation in the remaining 46 cases (r, = 0.529, P = 0.0002). In 39 cases sufficient RNA was obtained to allow further filters to be prepared containing larger amounts of RNA. The results obtained in these filters showed high concordance with those obtained originally for these patients (r, = 0.915, P < O.OOOl), validating the results. Our results suggest that ER expression in primary breast cancer is determined either by transcriptional modulation or by increased transcript stability.
INTRODUCTION
may partly explain this anomaly.’ Nevertheless, it is clear that, at present, ER expression is the best individual feature of a breast cancer available to us to predict likely response to endocrine therapy. Thus, it is essential that the molecular biological mechanisms that determine ER expression are fully investigated to enable future therapeutic interventions to be developed. Investigation of the ER gene has suggested that its alteration or amplification does not occur in primary human breast cancer.‘O Thus, changes in the ER gene would seem not to dictate ER expression. Quantitative comparison of the ER gene transcript and its protein has been performed.‘1-13 Whilst some studies were hampered by small numbers,“.12 they did suggest a significant correlation between ERmRNA and its protein. Therefore, whilst firm conclusions are difficult to draw, it does seem that ER expression may be determined either by transcriptional modulation or by increased transcript stability in primary human breast cancer. To provide further information on the mechanisms responsible for ER expression, we have studied 61 cases of primary human breast cancer. The ERmRNA and protein levels were compared following quantification using RNA slot blotting and semi-quantification using immunocytochemistty respectively.
Oestrogens are essential mitogens in the growth anddevelopment of normal breast tissue’ and act via specific nuclear receptors (ERs).~ These receptors are members of a family of nuclear receptors which includes the receptors for other steroids, thyroid hormone, vitamin D, and retinoic acid.3 Oestrogens are also recognized to play an important role in the development and subsequent progression of breast cancer.4 Overall, some 70% of human breast cancers express ER,5 the presence of which predicts survival and the likely response to endocrine therapy. ER expression is associated with improved overall and disease-free survival6 In addition, whilst 50-60% of ER positive breast cancers will respond to measures designed to reduce the circulating levels of oestrogen or interfere with its function,’ only 10% or ER negative breast cancers will do SO.~These studies highlight a major anomaly, namely that up to 50% of ER positive breast cancers do not respond to endocrine manipulation. The presence of dominant-negative ER variants Address correspondence to: A. K. Sharma, Academic Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK
237
238 The Breast MATERIALS
AND METHODS
Samples were obtained from 61 patients undergoing mastectomy or wide local excision for primary breast cancer, none of whom had received any prior therapy. The samples were immediately frozen in liquid nitrogen and stored at -70°C until required for processing for RNA extraction or immunocytochemistry. Of the 61 patients (age range 35-80 years, mean 61.4 years), nine were premenopausal and 52 were postmenopausal (Table). In addition, RNA was extracted from the breast cancer cell line MCF-7 to provide controls to monitor inter-filter variation. ER immunocytochemical assay Samples were processed and stained as previously described14utilizing the H222 monoclonal antibody available in kit form (ERICA monoclonal, Abbott Laboratories, North Chicago, Ill., USA). Parallel control sections using normal rat IgG antiserum were run to check for non-specific staining. Inclusion of control slides of MCF-7 cells enabled inter-assay variations to be monitored. Sections were examined using an Olympus BH2 light microscope with a dual viewing attachment by two independent observers (AS nd JG) and scored by consensus according to the intensity of staining and the proportion of cells stained. Scores of O-3 were allocated where by 0 = no staining; 1 = weak nuclear staining; 2 = moderate nuclear staining; and 3 = strong nuclear staining. A percentage estimation of cancer cells stained was made for each category and the final result, expressed as intensity of staining (I&, was obtained by the formula: c&J =
(%of1x1)+(%of2x2)+(%of3x3)
RNA slotting buffer (Amersham, UK) were applied to nylon filters (Hybond N, Amersham, UK) presoaked in 20 x standard sodium citrate (SSC) using a commercial slot blot apparatus (Schleicher & Schuell, Germany). In 39 cases sufficient RNA was obtained to allow a second series of filters containing 20 pg, 10 yg, and 5 pg of total RNA to be prepared. All filters contained slots with the appropriate quantities of MCF-7 cell RNA to allow inter filter variations to be monitored. RNA was fixed by baking the filters at 80°C for 10 min followed by exposure to ultraviolet light at 302 nm for 5 min. Filters were prehybridized at 45’C for 2 h in a hybridization oven (Techne Hybridiser HB-lD, Scotlab, UK) using 10 ml of a solution comprising 6.25 ml of 20 x standard sodium phosphate ethylenediaminetetraacetic acid, 1.25 ml of 100 x Denhardts solution (2% (w/v) each of polyvinylpyrollidone, ficoll-400 and bovine serum albumin), 0.625 ml of 20 x sodium dodecyl sulphate (SDS), 12.5 ml of formamide, 100 pl denatured sonicated herring sperm deoxyribonucleic acid (DNA) and 4.275 ml of double distilled water. The filters were then hybridized at 45°C for 16 h using the same solution containing radioactive labelled probe at 5 x lo6 dpm/ml. Following hybridization, the filters were washed twice at 45°C in 2 x SSC/O.l% SDS for 30 min each then in 0.2 x SSC/O.l% SDS at 45°C for 20 min. Autoradiographs were established by exposing the filters to Fuji Medical X-ray film with intensifying screens at -70°C for ERmRNA and room temperature for actin mRNA. Filters containing 14 pg of RNA required exposure for 14 days for ERmRNA and 12 h for actin mRNA. In filters containing 5-20 yg of RNA the exposure times were 7 days and 3 h respectively.
100
Frozen section samples of breast cancer were also stained with haematoxylin and eosin to confirm the presence of breast cancer and to determine histological assessability. Isolation of RNA Total cellular RNA was isolated using the guanidinium isothiocyanate method of extraction followed by centrifugation against a caesium chloride gradient.15Quantification of yields and their purity were assessedby spectrophotometry. The quality of RNA was verified by the integrity of the 28 and 18s ribosomal bands following agarose gel electrophoresis of 10 pg of total RNA (Fig. 1). RNA slot blotting Serial dilutions of 4 pg, 2 pg, and 1 pg of total RNA in
Probes The 1.8 kilobase (kb) cDNA HE0 probe was used to investigate ERmRNA levels. This contains the entire open reading frame of the human ER and includes 12 nucleotides upstream of the ATG codon and 11 nucleotides downstream of the TGA stop codon.16The genomic DNA pHA 4.1 probe” for actin was used to provide a control for variations in loading as actin is assumed to be equally expressed in all cells. la Labelling of the probes was achieved by the use of reagents obtainable in kit form (Prime-a-GeneR System, Promega Corporation, Madison, USA) employing the random primer method.lg Only reactions with greater than 30% (a-3*p) dCTP incorporation as measured by Cerenkov counting in the 3H channel on the 1215 Rackbeta II, liquid scintillation counter (Wallac Oy, Finland) were used immediately in the subsequent hybridization reactions.
Mechanisms responsible for ER expression in breast cancer 239
Table Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 41 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Patient details and results obtained following Age (ye@
Menopausal status
35 43 43 44 45 45 46 46 47 50 51 52 53 54 55 56 56 57 57 58 59 59 59 59 59 59 60 60 60 61 61 62 62 62 62 63 63 64 64 66 67 61 67 68 69 IO 70 71 71 71 72 72 73 74 74 77 78 78 79 79 80
Pre Pre Pre Pre Pre Pre Pre Pre Post Post Post Pre Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post
AU, arbitrary units; nd, not done; -, negative.
immunocytochemistry
ER ‘(EN 1.20
and RNA slot blotting
ERmRNA (l-4 pg. AU)
ERmRNA (5-20 pg, AU)
0.283 0.019
0.237 nd nd
0.05
0.001
0.32 0.25
0.125 0.196 0.067 0.049
0.186 nd
0.212 0.028 0.207 0.073 0.301 0.296 0.078 0.022 0.129 0.080 0.213 0.383 0.095 0.262 0.033
nd nd 0.166 0.470 0.259 nd 0.089 nd 0.120 nd nd nd 0.326 nd
0.154 0.449 0.332 0.343 0.172 0.246 0.167
0.174 nd 0.730 0.729 0.668 nd
0.054 0.061 0.149 0.139 0.054 0.184 0.707 0.215 0.018 0.356 0.625 0.119 0.195 0.585 0.53 1
0.164 nd nd nd 0.105 nd nd 0.606 nd 0.142 0.177 1.020 0.182 nd 0.463 0.621
0.139 0.109 -
0.231 0.250 -
0.70
0.90 0.10 1.80 0.65 0.40 0.75 0.70 0.60 0.70 0.05 0.80 0.20
0.80 1.65 1.10 1.60 0.20 1.10 1.15 0.80 0.70 0.20 0.70 1.10 0.90 1.25 1.00 0.60 0.90 0.38 0.90 0.10 1.30 1.00 0.50 -
nd
nd
240 The Breast cases (65.5%) following the ERICA (Table). Marked heterogeneity was observed between cases with the proportion of cells displaying any positivity ranging from 5% to 80%. In addition, intratumoural heterogeneity was observed with large variations in the intensity of staining between cancer cells in an individual patient. No binding was observed in the cytoplasm of tumour cells, in stromal components or in blood vessels or infiltrating macrophages.
6.2 Kb 28s
RNA slot blots
18s
MCF-7 c&l RNA
6.2 Kb 28s 1
2
3
4
5
6
Fig. 1 Thd specificity of the HE0 cDNA probe was confirmed by the finding of a 6.2 kb band following agarose gel electrophoresis of 10 pg of total RNA in both MCF-7 cells and in primary breast cancer samples. Lanes 5 and 6 demonstrate the presence of ERmRNA while lanes 14 demonstrate its absence in patients with primary breast cancer.
The specificity of the HE0 probe was confirmed by the finding of a 6.2 kb band following agarose gel electrophoresis of 10 Fg of total RNA in both MCF-7 cells and primary breast cancer samples (Fig. 1). Assessable results were obtained in all 61 cases where aliquots of 1-4 1.18of RNA were loaded (Fig. 2). ERmRNA was detected in 46 cases (75.4%, Table). In 40 cases (65.5%) both ERmRNA and ER protein were detected, in 6 cases ERmRNA but no ER protein was detected, and in 15 cases neither were detected. No cases were found to contain ER protein without ERmRNA. ERmRNA was found to be significantly correlated to ER protein (rs = 0.753, P < 0.0001, Fig. 3). Removal from the analysis of the 15 cases where neither ERmRNA or its protein were detected reduces the correlation but this is still highly significant (rs = 0.529, P = 0.0002). Similarly, assessable results were obtained in all 39 cases where sufficient RNA was available to enable filters to be prepared containing 5-20 l.tg of RNA. ERmRNA was detected in 23 cases (59%). In 21 cases (53.8%) both ERmRNA and ER protein were detected,
Quantification of mRNA Autoradiographic signals were quantified using a scanning densitometer (Model 620 Videodensitometer with Bio-Rad 1-D Analyst (1987) Software, Bio-Rad, UK). The three serial dilution signals for each receptor in each individual patient were divided by the actin signals. The final expression was determined by taking the mean of the three serial dilution ratios and expressed as arbitrary units (AU). Statistical analysis The relationship between ERmRNA and its protein was examined by Spearman’s rank correlation test (rJ. RESULTS ERICA Specific nuclear ER staining was immunolocalized in 40
4l-Q
2PS
Fig. 2 An example of the results obtained localization of slot blots containing l-4 pg used in rows 1 and 2 were found to contain whilst those in rows 3-6 contained varying
1cLg
using HE0 cDNA probe of total RNA. Patient samples no demonstratable ERmRNA amounts of ERmRNA.
Mechanisms responsible for ER expression in breast cancer 241 2.0 -
2.0 2.0
.
1.8 -
1.8 1.8.
.
1.6 -
1
l
1.6 -
1.4 -
. 1.2 -
.
.
.
. -
.
0.6
-*
. .
.
”
. .
.
l
.
.
. ..
1.0 0.8
.
.
.
..
l
.
rI = 0.753
.
0.1
0.2
0.3 0.4
0.5
0.6
P -c 0.0001
0.7
0.8
0.8 . 0.7 -
t)) 'i o 2 g
.
0
rs = 0.784
.
0.2 1~1
I
I
I
P < 0.0001 I
I
I
I
l
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
ERmRNA (AU)
levels obtained when l-4 kg of total RNA was used revealed a highly significant correlation. Even when the 15 ‘double-negative’ cases were excluded from the analysis this correlation was still highly significant (rs = 0.529, P = 0.0002).
. 0.6 -
. .
. '6 16 V-5
I1
Fig. 3 Comparison of ER protein levels with the results of ERmRNA
s.r
.
l .*
ERmRNA (AU)
Y s u) b s Y-
l
. -
0.4 -
.
o+ -r , I -1 I I 150
:-
-
.
.
.
0.8 0.8-
.
0.6
.
-
%
.
.
.
0.2
l.O-
.
l
0.4 -
I=
l
Fig. 5 The highly significant relationship between ERmRNA and its protein is contirmed from the results obtained using filters containing 5-20 Fg of total RNA.
in 2 cases (5.1%) ERmRNA alone was detected, and in 16 cases (41%) neither were detected. The results obtained from these filters show high concordance with the results obtained from the same cases in the l-4 pg filters (rS = 0.915, PC 0.0001, Fig. 4) thereby confirming our results. In these 39 cases ERmRNA was again demonstrated to be highly significantly correlated to ER protein (rS= 0.784, P < 0.0001, Fig. 5).
.
0.5 -
DISCUSSION
0.4 . 0.3 -
.*
.
. .
0.2 -
t
.
.
’ l **
E 0.1 1
r.
= 0.915
P < 0.0001
l . l
01%,
.
0.1I 0.2I 0.3,I
0.4 0.5 I 0.6 I 0.7 I 0.8 I 0.9I 1.0 I 1.1 I
ERmRNA (AU) Fig. 4 Comparison of the ERmRNA levels obtained in the 39 patients in whom filters containing l-4 Fg (y-axis) and S-20 pg (x-axis) were prepared reveals a highly significant correlation between the two series.
The proportion of primary human breast cancers found to be ER positive in the present study is compatible with previously published data.5 In addition, the heterogeneous nature of ER staining both between tumours and within a tumour is well documented.20Thus, the primary breast cancer specimens used in the present study seem to be of a similar ER profile to those used in previous investigations. The technique of RNA slot blotting provides a sensitive method for the detection and quantification of mRNA. It is possible to detect mRNAs that are present at approximately five copies per cell if 20 pg of total RNA is applied to a single slot. I5 Using this technique we have performed a quantitative analysis of the amount of mRNA coding for ER in 61 primary human breast cancer samples.
242 The Breast This analysis has allowed us to compare the level of specific mRNA with the level of ER protein measured using immunocytochemistry. The data presented in this study reveal a highly significant correlation between ERmRNA and its protein in vivo. Results derived from the examination of the ER gene. reveal that this is remarkably well conserved without any evidence of rearrangement or amplification either in vitrozl or in vivo.‘“,22 These studies suggest that changes in ER expression or function do not arise as a consequence of gross alterations in the ER gene. The findings presented in the current study would suggest that ER expression in primary human breast cancer is determined either by transcriptional modulation or by increased ER gene transcript stability. This observation is in broad agreement with previous studies using clinical breast cancer specimenV3 and with those using cell lines.23Further studies have revealed that ERmRNA and its protein both decrease when breast cancer cell lines are treated with progestin.” Whilst this would favour the concept of transcriptional modulation determining ER expression the possibility that progestin may directly or indirectly influence ERmRNA stability cannot be discounted. The demonstration of a highly significant correlation between ERmRNA and its protein reported in this study suggeststhat translational events contribute little to final ER expression. However, closer scrutiny of the results reveals that there may be up to a lo-fold spread in protein for a given mRNA level. RNA slot blotting involves homogenization of tissue thereby not allowing an assessment of the relative contributions of normal or benign components to be made. This problem is not encountered during immunocytochemical analysis as the tissue is directly visualised and only the malignant component assessed. This may account for our observations as normal or benign breast tissues contain less ER than malignant breast tissues*O and could thus dilute the level of ERmRNA. An alternative explanation is based on the concept of tumour heterogeneity which is well documented in human breast cancer.“) Thus, whilst the frozen section sample of breast cancer may have been taken from a relatively ER rich area the homogenized tissue used in RNA slot blotting may have been from an ER poor area or vice versa. The presence of dominant-positive or dominant-negative ERsg poses problems in assessing the likely endocrine responsiveness of human breast cancers. Nevertheless, ER expression still remains the best indicator. Devising new therapies to modulate ER expression and, therefore, endocrine responsiveness may thus need to target ER gene transcription. However, before embarking on this we need to elucidate further the precise mechanisms involved in determining or altering ER expression. In this light we are currently investigating ERmRNA and ER protein levels
in a series of patients before, during and at the time of relapse from endocrine therapy.
References 1. Laron Z, Kauli R, Pertzelan A. Clinical evidence on the role of oestrogens in the development of the breasts. Proc Sot Edinburgh 1989; 95B: 13-22. 2. Gorski J, Welshons W V, Sakai D et al. Evolution of a model of oestrogen action. Ret Prog Horm Res 1986; 2: 297-329. 3. Petkovich M, Brand N J, Krust A, Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature 1987; 330: 444-450. 4. Seibert K, Lippman M. Hormone receptors in breast cancer 1. Clinics in oncology. Eastboume: Saunders, 1982, 735-794. 5. Kvinnsland S. Steroid recentor assav and orormosis. Breast cancer treatment and prognosis. Oxford: Blackwell Scientific Publications, 1986, 140-154. 6 Harris A L, Nicholson S, Sainbury J R C, Farndon .I, Wright C. Epidermal growth factor receptors in breast cancer: association with early relapse and death, poor response to hormones and interactions with neu. .I Steroid Biochem 1989; 34: 123-131. 7 Osborne C K, Yochmowitz M G, McGuire W A, McGuire W L. The value of estrogen and progesterone receptors in the treatment of breast cancer. Cancer 1980; 46: 2884-2888. 8. Williams M R, Todd J H, Ellis I 0 et al. Oestrogen receptors in primary and advanced breast cancer: an eight year review of 704 cases. Br J Cancer 1987; 55: 67-73. 9. McGuire W L, Chamness G C, Fuqua S A W. Abnormal estrogen receptor in clinical breast cancer. J Steroid Biochem Mol Biol 1992; 43: 243-247. 10 Sharma A K, Grimshaw D, Horgan K, Douglas-Jones A G, Gee J M W, Nicholson R I. Analysis of the genes for oestrogen and epidermal growth factor receptors in human breast cancer. Br J Cancer 1995 (submitted). 11. Barrett-Lee P J, Travers M T, McClelland R A, Luqmani Y, Coombes R C. Characterization of estrogen receptor messenger RNA in human breast cancer. Cancer Res 1987; 47: 66536659. 12. Henry J A, Nicholson S, Farndon J R, Westley B R, May FEB. Measurement of oestrogen receptor mRNA levels in human breast tumours. Br J Cancer 1988; 58: 600-605. 13. May E, Mouriesse H, May-Levin F, Contesso G, Delarue J-C. A new approach allowing an early prognosis in breast cancer: the ratio of estrogen receptor (ER) ligand binding activity to the ER-specific mRNA level. Oncogene 1989; 4: 1037-1042. 14. Sharma A K, Horgan K, Douglas-Jones A, McClelland R, Gee J M W, Nicholson R. Dual immunocytochemical analysis of oestrogen and epidermal growth factor receptors in human breast cancer. Br J Cancer 1994; 69: 1032-1037. 15. Sambrook J, Fritsch E F, Maniatis T. Molecular cloning, 2nd edn. New York: Cold Spring Harbor Laboratory Press 1989. 16. Kumar V, Green S, Staub A, Chambon P. Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J 1986; 5: 2231-2236. 17. Khalili K. Salas C. Weimuenn R. Isolation and characterisation of humanactin genes cloned in phage lamda vectors. Gene 1983; 21: 9-17. 18. Gutrin M, Gabillot M, Mathieu M-C et al. Structure and expression of C-erbB-2 and EGF receptor genes in inflammatory and noninflammatory breast cancer: prognostic significance. Int J Cancer 1989; 43: 201-208. 19. Feinberg A P, Vogelstein B. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 1983; 132: 6-13. 20. Walker K 3, McClelland R A, Candlish W, Blarney R W, Nicholson R I. Heterogeneity of oestrogen receptor expression in normal and malignant breast tissue. Eur J Cancer 1992; 28: 3&37. 21. Watts C K W, Handel M L, King R J B, Sutherland R L. Oestrogen I
.
1
Mechanisms responsible for ER expression in breast cancer 243 receptor gene structure and function in breast cancer. J Steroid Biochem Mol Biol 1992; 41: 529-536. 22. Koh E H, Ro J, Wildrick D M, Hortobagyi G N, Blick M. Analysis of the oestrogen receptor gene structure in human breast cancer. Anticancer Res 1989; 9: 1841-1846. 23. Lee C S L, Hall R E, Alexander I E, Koga M, Shine J, Sutherland R L. Inverse relationship between estrogen receptor and epidermal growth factor receptor mRNA levels in human breast cancer cell lines. Growth Factors 1990: 3: 97-103.
24. Alexander I E, Shine J, Sutherland R L. Progestin regulation of estrogen receptor messenger RNA in human breast cancer cells. Mol Endocrinol 1990; 4: 821-828.
Date submitted 30 January 1995 Date accepted 5 June 1995
ORIGINAL ARTICLE
Mechanisms responsible for oestrogen receptor expression in primary human breast cancer A. K. Sharma, D. Grimshaw, K. Horgan, R. E. Mansel, J. M. W . Gee* and R. I. Nicholson* Department of Surgery and *Tenovus Cancer Research Centre, University of Wales College of Medicine, Cardifi
UK
S UMMA R Y. Oestrogen receptor (ER) status in human breast cancer provides the best parameter available at present to determine the likely response to endocrine therapy. However, the precise mechanisms responsible for ER expression have yet to be fully elucidated. In order to examine this we have quantitatively analysed the mRNA and protein levels of ER in 61 patients with primary breast cancer using the techniques of ribonucleic acid (RNA) slot blotting and immunocytochemistry. Using the H222 monoclonal antibody, ER staining was observed in 40 cases (65.5%) following immunocytochemical assay. RNA slot blotting using a complimentary deoxyribonucleic acid probe (HEO) revealed ERmRNA to be present in 46 cases (75.4%). The mRNA and protein levels of ER were found to be significantly correlated (rs = 0.753, P < 0.0001). Exclusion of 15 cases where neither mRNA nor protein was detected still revealed a significant correlation in the remaining 46 cases (r, = 0.529, P = 0.0002). In 39 cases sufficient RNA was obtained to allow further filters to be prepared containing larger amounts of RNA. The results obtained in these filters showed high concordance with those obtained originally for these patients (r, = 0.915, P < O.OOOl), validating the results. Our results suggest that ER expression in primary breast cancer is determined either by transcriptional modulation or by increased transcript stability.
INTRODUCTION
may partly explain this anomaly.’ Nevertheless, it is clear that, at present, ER expression is the best individual feature of a breast cancer available to us to predict likely response to endocrine therapy. Thus, it is essential that the molecular biological mechanisms that determine ER expression are fully investigated to enable future therapeutic interventions to be developed. Investigation of the ER gene has suggested that its alteration or amplification does not occur in primary human breast cancer.‘O Thus, changes in the ER gene would seem not to dictate ER expression. Quantitative comparison of the ER gene transcript and its protein has been performed.‘1-13 Whilst some studies were hampered by small numbers,“.12 they did suggest a significant correlation between ERmRNA and its protein. Therefore, whilst firm conclusions are difficult to draw, it does seem that ER expression may be determined either by transcriptional modulation or by increased transcript stability in primary human breast cancer. To provide further information on the mechanisms responsible for ER expression, we have studied 61 cases of primary human breast cancer. The ERmRNA and protein levels were compared following quantification using RNA slot blotting and semi-quantification using immunocytochemistty respectively.
Oestrogens are essential mitogens in the growth anddevelopment of normal breast tissue’ and act via specific nuclear receptors (ERs).~ These receptors are members of a family of nuclear receptors which includes the receptors for other steroids, thyroid hormone, vitamin D, and retinoic acid.3 Oestrogens are also recognized to play an important role in the development and subsequent progression of breast cancer.4 Overall, some 70% of human breast cancers express ER,5 the presence of which predicts survival and the likely response to endocrine therapy. ER expression is associated with improved overall and disease-free survival6 In addition, whilst 50-60% of ER positive breast cancers will respond to measures designed to reduce the circulating levels of oestrogen or interfere with its function,’ only 10% or ER negative breast cancers will do SO.~These studies highlight a major anomaly, namely that up to 50% of ER positive breast cancers do not respond to endocrine manipulation. The presence of dominant-negative ER variants Address correspondence to: A. K. Sharma, Academic Department of Surgery, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, UK
237
238 The Breast MATERIALS
AND METHODS
Samples were obtained from 61 patients undergoing mastectomy or wide local excision for primary breast cancer, none of whom had received any prior therapy. The samples were immediately frozen in liquid nitrogen and stored at -70°C until required for processing for RNA extraction or immunocytochemistry. Of the 61 patients (age range 35-80 years, mean 61.4 years), nine were premenopausal and 52 were postmenopausal (Table). In addition, RNA was extracted from the breast cancer cell line MCF-7 to provide controls to monitor inter-filter variation. ER immunocytochemical assay Samples were processed and stained as previously described14utilizing the H222 monoclonal antibody available in kit form (ERICA monoclonal, Abbott Laboratories, North Chicago, Ill., USA). Parallel control sections using normal rat IgG antiserum were run to check for non-specific staining. Inclusion of control slides of MCF-7 cells enabled inter-assay variations to be monitored. Sections were examined using an Olympus BH2 light microscope with a dual viewing attachment by two independent observers (AS nd JG) and scored by consensus according to the intensity of staining and the proportion of cells stained. Scores of O-3 were allocated where by 0 = no staining; 1 = weak nuclear staining; 2 = moderate nuclear staining; and 3 = strong nuclear staining. A percentage estimation of cancer cells stained was made for each category and the final result, expressed as intensity of staining (I&, was obtained by the formula: c&J =
(%of1x1)+(%of2x2)+(%of3x3)
RNA slotting buffer (Amersham, UK) were applied to nylon filters (Hybond N, Amersham, UK) presoaked in 20 x standard sodium citrate (SSC) using a commercial slot blot apparatus (Schleicher & Schuell, Germany). In 39 cases sufficient RNA was obtained to allow a second series of filters containing 20 pg, 10 yg, and 5 pg of total RNA to be prepared. All filters contained slots with the appropriate quantities of MCF-7 cell RNA to allow inter filter variations to be monitored. RNA was fixed by baking the filters at 80°C for 10 min followed by exposure to ultraviolet light at 302 nm for 5 min. Filters were prehybridized at 45’C for 2 h in a hybridization oven (Techne Hybridiser HB-lD, Scotlab, UK) using 10 ml of a solution comprising 6.25 ml of 20 x standard sodium phosphate ethylenediaminetetraacetic acid, 1.25 ml of 100 x Denhardts solution (2% (w/v) each of polyvinylpyrollidone, ficoll-400 and bovine serum albumin), 0.625 ml of 20 x sodium dodecyl sulphate (SDS), 12.5 ml of formamide, 100 pl denatured sonicated herring sperm deoxyribonucleic acid (DNA) and 4.275 ml of double distilled water. The filters were then hybridized at 45°C for 16 h using the same solution containing radioactive labelled probe at 5 x lo6 dpm/ml. Following hybridization, the filters were washed twice at 45°C in 2 x SSC/O.l% SDS for 30 min each then in 0.2 x SSC/O.l% SDS at 45°C for 20 min. Autoradiographs were established by exposing the filters to Fuji Medical X-ray film with intensifying screens at -70°C for ERmRNA and room temperature for actin mRNA. Filters containing 14 pg of RNA required exposure for 14 days for ERmRNA and 12 h for actin mRNA. In filters containing 5-20 yg of RNA the exposure times were 7 days and 3 h respectively.
100
Frozen section samples of breast cancer were also stained with haematoxylin and eosin to confirm the presence of breast cancer and to determine histological assessability. Isolation of RNA Total cellular RNA was isolated using the guanidinium isothiocyanate method of extraction followed by centrifugation against a caesium chloride gradient.15Quantification of yields and their purity were assessedby spectrophotometry. The quality of RNA was verified by the integrity of the 28 and 18s ribosomal bands following agarose gel electrophoresis of 10 pg of total RNA (Fig. 1). RNA slot blotting Serial dilutions of 4 pg, 2 pg, and 1 pg of total RNA in
Probes The 1.8 kilobase (kb) cDNA HE0 probe was used to investigate ERmRNA levels. This contains the entire open reading frame of the human ER and includes 12 nucleotides upstream of the ATG codon and 11 nucleotides downstream of the TGA stop codon.16The genomic DNA pHA 4.1 probe” for actin was used to provide a control for variations in loading as actin is assumed to be equally expressed in all cells. la Labelling of the probes was achieved by the use of reagents obtainable in kit form (Prime-a-GeneR System, Promega Corporation, Madison, USA) employing the random primer method.lg Only reactions with greater than 30% (a-3*p) dCTP incorporation as measured by Cerenkov counting in the 3H channel on the 1215 Rackbeta II, liquid scintillation counter (Wallac Oy, Finland) were used immediately in the subsequent hybridization reactions.
Mechanisms responsible for ER expression in breast cancer 239
Table Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 41 48 49 50 51 52 53 54 55 56 57 58 59 60 61
Patient details and results obtained following Age (ye@
Menopausal status
35 43 43 44 45 45 46 46 47 50 51 52 53 54 55 56 56 57 57 58 59 59 59 59 59 59 60 60 60 61 61 62 62 62 62 63 63 64 64 66 67 61 67 68 69 IO 70 71 71 71 72 72 73 74 74 77 78 78 79 79 80
Pre Pre Pre Pre Pre Pre Pre Pre Post Post Post Pre Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post Post
AU, arbitrary units; nd, not done; -, negative.
immunocytochemistry
ER ‘(EN 1.20
and RNA slot blotting
ERmRNA (l-4 pg. AU)
ERmRNA (5-20 pg, AU)
0.283 0.019
0.237 nd nd
0.05
0.001
0.32 0.25
0.125 0.196 0.067 0.049
0.186 nd
0.212 0.028 0.207 0.073 0.301 0.296 0.078 0.022 0.129 0.080 0.213 0.383 0.095 0.262 0.033
nd nd 0.166 0.470 0.259 nd 0.089 nd 0.120 nd nd nd 0.326 nd
0.154 0.449 0.332 0.343 0.172 0.246 0.167
0.174 nd 0.730 0.729 0.668 nd
0.054 0.061 0.149 0.139 0.054 0.184 0.707 0.215 0.018 0.356 0.625 0.119 0.195 0.585 0.53 1
0.164 nd nd nd 0.105 nd nd 0.606 nd 0.142 0.177 1.020 0.182 nd 0.463 0.621
0.139 0.109 -
0.231 0.250 -
0.70
0.90 0.10 1.80 0.65 0.40 0.75 0.70 0.60 0.70 0.05 0.80 0.20
0.80 1.65 1.10 1.60 0.20 1.10 1.15 0.80 0.70 0.20 0.70 1.10 0.90 1.25 1.00 0.60 0.90 0.38 0.90 0.10 1.30 1.00 0.50 -
nd
nd
240 The Breast cases (65.5%) following the ERICA (Table). Marked heterogeneity was observed between cases with the proportion of cells displaying any positivity ranging from 5% to 80%. In addition, intratumoural heterogeneity was observed with large variations in the intensity of staining between cancer cells in an individual patient. No binding was observed in the cytoplasm of tumour cells, in stromal components or in blood vessels or infiltrating macrophages.
6.2 Kb 28s
RNA slot blots
18s
MCF-7 c&l RNA
6.2 Kb 28s 1
2
3
4
5
6
Fig. 1 Thd specificity of the HE0 cDNA probe was confirmed by the finding of a 6.2 kb band following agarose gel electrophoresis of 10 pg of total RNA in both MCF-7 cells and in primary breast cancer samples. Lanes 5 and 6 demonstrate the presence of ERmRNA while lanes 14 demonstrate its absence in patients with primary breast cancer.
The specificity of the HE0 probe was confirmed by the finding of a 6.2 kb band following agarose gel electrophoresis of 10 Fg of total RNA in both MCF-7 cells and primary breast cancer samples (Fig. 1). Assessable results were obtained in all 61 cases where aliquots of 1-4 1.18of RNA were loaded (Fig. 2). ERmRNA was detected in 46 cases (75.4%, Table). In 40 cases (65.5%) both ERmRNA and ER protein were detected, in 6 cases ERmRNA but no ER protein was detected, and in 15 cases neither were detected. No cases were found to contain ER protein without ERmRNA. ERmRNA was found to be significantly correlated to ER protein (rs = 0.753, P < 0.0001, Fig. 3). Removal from the analysis of the 15 cases where neither ERmRNA or its protein were detected reduces the correlation but this is still highly significant (rs = 0.529, P = 0.0002). Similarly, assessable results were obtained in all 39 cases where sufficient RNA was available to enable filters to be prepared containing 5-20 l.tg of RNA. ERmRNA was detected in 23 cases (59%). In 21 cases (53.8%) both ERmRNA and ER protein were detected,
Quantification of mRNA Autoradiographic signals were quantified using a scanning densitometer (Model 620 Videodensitometer with Bio-Rad 1-D Analyst (1987) Software, Bio-Rad, UK). The three serial dilution signals for each receptor in each individual patient were divided by the actin signals. The final expression was determined by taking the mean of the three serial dilution ratios and expressed as arbitrary units (AU). Statistical analysis The relationship between ERmRNA and its protein was examined by Spearman’s rank correlation test (rJ. RESULTS ERICA Specific nuclear ER staining was immunolocalized in 40
4l-Q
2PS
Fig. 2 An example of the results obtained localization of slot blots containing l-4 pg used in rows 1 and 2 were found to contain whilst those in rows 3-6 contained varying
1cLg
using HE0 cDNA probe of total RNA. Patient samples no demonstratable ERmRNA amounts of ERmRNA.
Mechanisms responsible for ER expression in breast cancer 241 2.0 -
2.0 2.0
.
1.8 -
1.8 1.8.
.
1.6 -
1
l
1.6 -
1.4 -
. 1.2 -
.
.
.
. -
.
0.6
-*
. .
.
”
. .
.
l
.
.
. ..
1.0 0.8
.
.
.
..
l
.
rI = 0.753
.
0.1
0.2
0.3 0.4
0.5
0.6
P -c 0.0001
0.7
0.8
0.8 . 0.7 -
t)) 'i o 2 g
.
0
rs = 0.784
.
0.2 1~1
I
I
I
P < 0.0001 I
I
I
I
l
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
ERmRNA (AU)
levels obtained when l-4 kg of total RNA was used revealed a highly significant correlation. Even when the 15 ‘double-negative’ cases were excluded from the analysis this correlation was still highly significant (rs = 0.529, P = 0.0002).
. 0.6 -
. .
. '6 16 V-5
I1
Fig. 3 Comparison of ER protein levels with the results of ERmRNA
s.r
.
l .*
ERmRNA (AU)
Y s u) b s Y-
l
. -
0.4 -
.
o+ -r , I -1 I I 150
:-
-
.
.
.
0.8 0.8-
.
0.6
.
-
%
.
.
.
0.2
l.O-
.
l
0.4 -
I=
l
Fig. 5 The highly significant relationship between ERmRNA and its protein is contirmed from the results obtained using filters containing 5-20 Fg of total RNA.
in 2 cases (5.1%) ERmRNA alone was detected, and in 16 cases (41%) neither were detected. The results obtained from these filters show high concordance with the results obtained from the same cases in the l-4 pg filters (rS = 0.915, PC 0.0001, Fig. 4) thereby confirming our results. In these 39 cases ERmRNA was again demonstrated to be highly significantly correlated to ER protein (rS= 0.784, P < 0.0001, Fig. 5).
.
0.5 -
DISCUSSION
0.4 . 0.3 -
.*
.
. .
0.2 -
t
.
.
’ l **
E 0.1 1
r.
= 0.915
P < 0.0001
l . l
01%,
.
0.1I 0.2I 0.3,I
0.4 0.5 I 0.6 I 0.7 I 0.8 I 0.9I 1.0 I 1.1 I
ERmRNA (AU) Fig. 4 Comparison of the ERmRNA levels obtained in the 39 patients in whom filters containing l-4 Fg (y-axis) and S-20 pg (x-axis) were prepared reveals a highly significant correlation between the two series.
The proportion of primary human breast cancers found to be ER positive in the present study is compatible with previously published data.5 In addition, the heterogeneous nature of ER staining both between tumours and within a tumour is well documented.20Thus, the primary breast cancer specimens used in the present study seem to be of a similar ER profile to those used in previous investigations. The technique of RNA slot blotting provides a sensitive method for the detection and quantification of mRNA. It is possible to detect mRNAs that are present at approximately five copies per cell if 20 pg of total RNA is applied to a single slot. I5 Using this technique we have performed a quantitative analysis of the amount of mRNA coding for ER in 61 primary human breast cancer samples.
242 The Breast This analysis has allowed us to compare the level of specific mRNA with the level of ER protein measured using immunocytochemistry. The data presented in this study reveal a highly significant correlation between ERmRNA and its protein in vivo. Results derived from the examination of the ER gene. reveal that this is remarkably well conserved without any evidence of rearrangement or amplification either in vitrozl or in vivo.‘“,22 These studies suggest that changes in ER expression or function do not arise as a consequence of gross alterations in the ER gene. The findings presented in the current study would suggest that ER expression in primary human breast cancer is determined either by transcriptional modulation or by increased ER gene transcript stability. This observation is in broad agreement with previous studies using clinical breast cancer specimenV3 and with those using cell lines.23Further studies have revealed that ERmRNA and its protein both decrease when breast cancer cell lines are treated with progestin.” Whilst this would favour the concept of transcriptional modulation determining ER expression the possibility that progestin may directly or indirectly influence ERmRNA stability cannot be discounted. The demonstration of a highly significant correlation between ERmRNA and its protein reported in this study suggeststhat translational events contribute little to final ER expression. However, closer scrutiny of the results reveals that there may be up to a lo-fold spread in protein for a given mRNA level. RNA slot blotting involves homogenization of tissue thereby not allowing an assessment of the relative contributions of normal or benign components to be made. This problem is not encountered during immunocytochemical analysis as the tissue is directly visualised and only the malignant component assessed. This may account for our observations as normal or benign breast tissues contain less ER than malignant breast tissues*O and could thus dilute the level of ERmRNA. An alternative explanation is based on the concept of tumour heterogeneity which is well documented in human breast cancer.“) Thus, whilst the frozen section sample of breast cancer may have been taken from a relatively ER rich area the homogenized tissue used in RNA slot blotting may have been from an ER poor area or vice versa. The presence of dominant-positive or dominant-negative ERsg poses problems in assessing the likely endocrine responsiveness of human breast cancers. Nevertheless, ER expression still remains the best indicator. Devising new therapies to modulate ER expression and, therefore, endocrine responsiveness may thus need to target ER gene transcription. However, before embarking on this we need to elucidate further the precise mechanisms involved in determining or altering ER expression. In this light we are currently investigating ERmRNA and ER protein levels
in a series of patients before, during and at the time of relapse from endocrine therapy.
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.
1
Mechanisms responsible for ER expression in breast cancer 243 receptor gene structure and function in breast cancer. J Steroid Biochem Mol Biol 1992; 41: 529-536. 22. Koh E H, Ro J, Wildrick D M, Hortobagyi G N, Blick M. Analysis of the oestrogen receptor gene structure in human breast cancer. Anticancer Res 1989; 9: 1841-1846. 23. Lee C S L, Hall R E, Alexander I E, Koga M, Shine J, Sutherland R L. Inverse relationship between estrogen receptor and epidermal growth factor receptor mRNA levels in human breast cancer cell lines. Growth Factors 1990: 3: 97-103.
24. Alexander I E, Shine J, Sutherland R L. Progestin regulation of estrogen receptor messenger RNA in human breast cancer cells. Mol Endocrinol 1990; 4: 821-828.
Date submitted 30 January 1995 Date accepted 5 June 1995